More than half of the 18,000 patients diagnosed with malignant primary brain tumors in the U.S. each year have glioblastoma (GBM), the most common and most aggressive primary malignant brain tumor in adults. Over the last two decades, the major breakthrough in the treatment for GBM has been the addition of the DNA alkylating agent temozolomide (TMZ) to the standard of care (surgery and radiation) yielding an increase in the median survival from 12.1 months to 14.6 months. Despite this advancement, 90% of GBM patients die within 5 years, a colossal failure attributed to TMZ resistance. One of the major predictor of GBM response to TMZ is the MGMT promoter methylation status. This enzyme removes the DNA adduct, induced by TMZ, leading to cell survival. Thus, patients whose tumors have transcriptional silencing of the MGMT gene, mediated by promoter methylation, are most likely to benefit from TMZ. Given that all glioblastomas recur to a tumor lesion with acquired resistance to TMZ, novel adjuvant therapies could be highly beneficial to GBM patients. Recently, it was shown that a subpopulation of tumor cells, called glioblastoma stem cells (GSCs or tumor initiating cells), are responsible for tumor recurrence and resistance to conventional therapies. In this proposal, we will use a multiplex secreted reporter system for high-throughput screening to find drugs that either synergizes with TMZ on GBM stem cells from both newly diagnosed and recurrent tumors, irrespective of the MGMT status, or that kills a specific GBM stem cells subtype. We will simultaneously screen in the same well for drugs which act on three different GBM stem cells subpopulations: (1) obtained from newly diagnosed tumors with methylated MGMT promoter; (2) newly diagnosed GBM with unmethylated MGMT promoter; (3) recurrent TMZ-resistant tumors. We will use primary GBM stem cells dissociated from patient tumors sections and grow them as neural spheres which maintain the phenotype/genotype of the original tumor. Dose- and time-dependent experiments of our drug hits will be performed and validated using secondary screening assays in culture. A set of analogues of the most promising hits will be purchased, if available commercially, or synthesized by medicinal chemistry to make these drugs fit-for-purpose, to facilitate their study in cells, and for pull down assays to find targets of drug hits. Finally, specificity of drug hits o glioblastoma will be tested using a panel of normal cells and stem cells in culture.